Asymmetric reduction of ethyl acetoacetate catalyzed by immobilized Acetobacter sp. CCTCC M209061 cells in hydrophilic ionic liquid hybrid systemBiotechnology and Bioprocess Engineering


Ping Wei, Pei Xu, Xiao-Ting Wang, Wen-Yong Lou, Min-Hua Zong
Applied Microbiology and Biotechnology / Biotechnology / Biomedical Engineering / Bioengineering


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Biotechnology and Bioprocess Engineering 20: 324-332 (2015)

DOI 10.1007/s12257-014-0751-8

Asymmetric Reduction of Ethyl Acetoacetate Catalyzed by

Immobilized Acetobacter sp. CCTCC M209061 Cells in Hydrophilic

Ionic Liquid Hybrid System

Ping Wei, Pei Xu, Xiao-Ting Wang, Wen-Yong Lou, and Min-Hua Zong

Received: 3 November 2014 / Revised: 24 December 2014 / Accepted: 1 January 2015 © The Korean Society for Biotechnology and Bioengineering and Springer 2015

Abstract The asymmetric reduction of ethyl acetoacetate (EAA) to ethyl (R)-3-hydroxybutyrate [(R)-EHB] using immobilized Acetobacter sp. CCTCC M209061 cells was successfully conducted in a hydrophilic ionic liquid (IL)containing system. The best one of all the tested watermiscible ILs was 1-(hydroxyethyl)-3-methylimidazolium hydrochloride (C2OHMIM·Cl). In C2OHMIM·Cl-aqueous buffer hybrid system, it was found that the optimal IL concentration, substrate and co-substrate concentration, reaction temperature, buffer pH and shaking rate were 0.5 mol/L, 45 mmol/L, 80 mmol/L, 35oC, pH 5.5 and 200 rpm, respectively. Under the optimized reaction conditions, the initial reaction rate, the yield and the product e.e. reached 4.90 µmol/min, 95.3 and > 99.0%, respectively, which were much higher than the corresponding values reported previously. The efficient biocatalytic process mediated by the immobilized cells was feasible on 500 mL preparative scale, and the biocatalysts showed good operational stability and could be recycled for at least 10 batches.

Keywords: Acetobacter sp. CCTCC M209061 cells, asymmetric reduction, hydrophilic ILs, immobilization, ethyl acetoacetate, ethyl (R)-3-hydroxybutyrate 1. Introduction

Hydroxyesters are important building blocks which have been widely used in the synthesis of varieties of important chiral pharmaceuticals, agrochemicals, liquid crystals and flavors [1]. Among them, enantiomerically pure ethyl 3hydroxybutyrate (EHB) is a versatile intermediate for the formation of various biologically and structurally interesting compounds and pharmaceuticals [1,2]. For example, ethyl (S)-3-hydroxybutyrate [(S)-EHB] was used as chiral intermediate for the preparation of lavandulol, sulcatol, and prenophorin, and ethyl (R)-3-hydroxybutyrate [(R)-EHB] acts as an important blocks for the synthesis of L-carnitine, imipenem and other carbapenems chiral drugs [3]. According to the literatures, the biocatalytic asymmetric reduction of EAA was mostly carried out in aqueous phase with (S)EHB as the dominant product [4,5]. To date, the biocatalytic synthesis of (R)-EHB has remained unexplored largely, with only few accounts [6-8]. In these cases, some microorganisms including Paracoccus denitrificans, Saccaromyces cerevisiae, Aspergilus niger, Kluyveromyces marxianus,

Hansenula sp. and Dekera sp. were able to catalyze the anti-Prelog stereoselective reduction of EAA to (R)-EHB, but afforded poor results in aqueous system (less than 88.7% product e.e.; 64% product yield; 104 h reaction time). Wang et al. [8] described the biocatalytic synthesis of (R)-EHB catalyzed by Pichia membranaefaciens Hansen cells in ionic liquid-containing system, and the yield (77.8%) of (R)-EHB and product e.e. (73.0%) obtained

Ping Wei, Pei Xu, Xiao-Ting Wang, Wen-Yong Lou*

Lab of Applied Biocatalysis, College of Light Industry and Food Sciences,

South China University of Technology, Guangzhou, Guangdong, China

Tel: +86-20-2223-6669; Fax: +86-20-2223-6669


Ping Wei, Min-Hua Zong*

School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou, Guangdong, China

Tel: +86-20-8711-1452; Fax: +86-20-2223-6669


Wen-Yong Lou, Min-Hua Zong

State Key Laboratory of Pulp and Paper Engineering, South China

University of Technology, Guangzhou, Guangdong, China


Asymmetric Reduction of Ethyl Acetoacetate Catalyzed by Immobilized Acetobacter sp. CCTCC M209061 Cells in Hydrophilic… 325 were unsatisfactory.

In our previous effort to improve the yield and the product e.e of asymmetric reduction of EAA to (R)-EHB, [9,10] immobilized Acetobacter sp. CCTCC M209061 cells were found to be capable of catalyzing the asymmetric reduction of EAA to (R)-EHB in aqueous system with a good yield (82.6%) and an excellent product e.e. (> 99.0%). In addition, using an IL/buffer biphasic system instead of the aqueous one could improve the reaction efficiency by increasing substrate concentration (35 mmol/

L vs 55 mmol/L) and yield (82.6% vs. 90.8%). As it has been reported that adding a small amount of ILs in aqueous systems could enhance the efficiency of whole-cell catalyzed asymmetric reductions [11].

In the present work, different kinds of hydrophilic ILs were added to aqueous system for examining the effects of hydrophilic ILs on immobilized Acetobacter sp. CCTCC

M209061 cell-mediated asymmetric reduction of EAA to (R)-EHB (Scheme 1). In this scheme, EAA is reduced to enantiopure (R)-EHB while converting NAD(P)H to

NAD(P)+, and glucose is simultaneously oxidized to CO2, presumably driving the reduction reaction by regenerating

NAD(P)H from NAD(P)+. Moreover, the efficient biocatalytic process with hydrophilic ILs for EAA reduction to (R)EHB was evaluated on a preparative scale. 2. Materials and Methods 2.1. Biological and chemical materials

Acetobacter sp.CCTCC M209061 was isolated from

Chinese kefir grains by our research group and preserved in our laboratory [12].

Ethyl acetoacetate (99% purity, EAA) was from Alfa

Aesar (USA). Ethyl 3-hydroxybutyrate (98% purity, EHB), ethyl (R)-3-hydroxybutyrate {98% purity, (R)-EHB} and n-decane (> 99% purity) were obtained from SigmaAldrich (USA). The twelve ILs used in this work, 1-(2'-hydroxyl)ethyl-3-methylimidazolium nitrate (C2OHMIM·NO3) 1-(2'hydroxyl)ethyl-3-methylimidazolium tetrafluoroborate (C2OHMIM·BF4) 1-(2'-hydroxyl)ethyl-3-methylimidazolium hexafluorophoshpate (C2OHMIM·PF6) 1-(2'-hydroxyl)ethyl3-methylimidazolium hydrochloride (C2OHMIM·Cl) 1-(2'hydroxylethyl)-3-methylimidazolium trifluoromethanesulfonate (C2OHMIM·TfO) 1-ethyl-3-methylimidazolium tetrafluoroborate (C2MIM·BF4) 1-propyl-3-methylimidazolium tetrafluoroborate (C3MIM·BF4) 1-butyl-3-methylimidazolium tetrafluoroborate (C4MIM·BF4) 1-butyl-3-methylimidazolium hydrochloride (C4MIM·Cl) 1-butyl-3methylimidazolium hydrobromide (C4MIM·Br) 1-pentyl3-methylimidazolium hydrobromide (C5MIM·Br) 1-hexyl3-methylimidazolium hydrobromide (C6MIM·Br) were purchased from Lanzhou Institute of Chemical Physics (China) with a purity of > 98%. All other chemicals were also from commercial sources and were of analytical grade. 2.2. Cultivation and immobilization of Acetobacter sp.